The present invention relates to the field of enhancing proteins, in particular that of proteins enhanced by molecular change. It pertains to a variant of a phytase, termed enhanced, in that it has better thermostability and/or activity compared with the original phytase. The invention also pertains to a nucleic acid coding for said variant, to a cassette or an expression vector containing said variant, to a host cell expressing said variant, to a composition comprising said variant, and also to the uses thereof, principally in the preparation of food additives and animal feed.
Phytate is the principal phosphorus storage compound in plants. This molecule, also known as phytic acid or inositol-hexa-phosphate (InsP6 or myo-inositol hexakisphosphate), consists of a cyclohexane to which six phosphate groups are bonded. The phytate represents approximately 70% of plant phosphate, the remaining 30% being present in the free form. Further, phosphate residues of phytate chelate divalent and trivalent cations such as calcium, iron, zinc, magnesium, copper, manganese and molybdenum, which are essential for nutrition.
Phytases are enzymes that hydrolyze phytate: such enzymes naturally release one or two phosphates, rarely three, which can then be adsorbed into the digestive system; other reaction products are rarely inositol-tri-phosphate, principally inositol tetra- and penta-phosphate (FIG. 1). Phytases constitute a family of enzymes that is widely represented in nature: many organisms, from the bacterium to the plant via fungi and certain animals, express one or more of them. However, mammals do not express any; ruminants or polygastric animals such as cattle and horses have endogenous microorganisms in their gastrointestinal tract that can degrade the phytate, but this does not apply with monogastric animals, principally pigs and poultry, for example, and so the phytate originating from ingested plants does not represent a useful source of phosphorus. Thus, it is necessary to add free phosphates to their feed. However, a portion of those added free phosphates and practically all of the phytate ingested by the animals are discharged into the environment. The phytate is then degraded by bacteria in the ground and ends up in the ground water and rivers in the form of free phosphate. In regions with a high concentration of stock farming, such agro-industrial phosphate waste, in the free form or in the form of phytate, represents a major source of pollution, which in particular results in the proliferation of green algae in rivers and watercourses. In addition to the esthetic aspect, such algae have a major ecological impact since they compete with local plant species, in particular as regards consumption of dissolved oxygen.
One way of being able to use phosphate stored in the form of phytate as a source of phosphate by mammals is to introduce exogenous phytase into food. Adding said enzyme thus represents an alternative to using inorganic phosphate as a food supplement. Further, by rendering the phosphate of the phytate accessible, they can also provide better accessibility to metallic ions chelated onto the phosphate of the phytate, as well as to proteins bonded to the phytate, rendering them nutritionally available. Phytases are currently used relatively systematically in animal feed. They are used as a partial replacement for phosphates and they also render proteins, amino acids, and calcium more accessible.
However, there are several limitations to using phytases in animal feed:                the insufficient effectiveness of phytases in releasing phosphates constitutes a first limitation. Phosphates are still added to animal feed, even in the presence of exogenous phytases. However, if all of the phytate were to be converted into free phosphates, supplements would no longer be required; thus, there is a genuine need to propose more active phytases;        the majority of enzymes in current use cannot be added directly to feed, since they cannot withstand the granulation process, which involves heating the feed to 95° C. for 90 seconds. The enzymes are thus sprayed onto the feed after the granulation stage, which represents an additional cost and constitutes a second limitation; thus, there is great demand for the availability of more thermostable phytases.        
The aim of the present invention is to overcome current limitations by generating a phytase that is sufficiently active to have a substantial impact on the need for supplementing with phosphates and is sufficiently thermostable to be able to be added directly to the feed of animals, without having to use a spraying technique.
Many phytases have featured in publications and patent applications, or are even already in use in agro-industrial applications. However, none of them have been able to dispense with the need for supplementing stock farm animal feed with phosphates, and none of them is sufficiently thermostable to be added directly to animal feed without having to use a spraying technique.
The majority of publications pertain to phytases from Aspergillus niger and Escherichia coli. Other phytases of microbial origin or deriving from plants have also been studied, the problem being to obtain thermostable phytases having a specific activity that is greater than that of Aspergillus niger (250 U/mg). The phytases described in the literature and the patent applications deal with fungi (basidiomycetes and ascomycetes), yeasts and bacteria.
Many phytases have been isolated from fungi and derive in the main from the Aspergillus family, but from Absidia, Acrophiaiophora, Agrocybe, Calcarisporiella, Cheatomium, Corynascus, Mucor, Mycelia, Myriococcurn, Penicillium, Peniophora, Rhizomucor, Rhizopus or even from Trametes, Sporotrichum, Neurospora, Trichoderma, Cladosporium, Myceiiophthora, Taleromyces, Thielavia, Humicola, Paxillus and Thermoascus. 
A great deal of information is available regarding the specific activities and Km of such enzymes, such as that in Wyss et al 1999 (Appl. Environ. Microbiol. 65 (2) 367-373). Overall, Aspergillus phytases are characterized by high Km values of 5 μM to 20 μM, an optimum temperature of 57° C. and an optimum pH of 5.5 except for Aspergillus fumigatus (opt pH.=6), but have very low specific activities ranging from 100 to 250 U/mg.
Certain phytases deriving from other fungi have exhibited interesting properties, in particular high specific activities, such as the phytase from Trametes pubescens or the phytase from Peniophora lycii (respectively 1200 U/mg and 1080 U/mg in WO 98/28408). Cladosporium sp. has an interesting phytase with a specific activity of 900 U/mg and a Km of 15.2 μM, but has a low optimum temperature of 40° C. In addition, the 6-phytase from Ceriporia sp. has a specific activity of 11040 U/mg. Some complementary information was published by Lassen et al, 2001 (Appl. Environ. Microbiol. 67 (10) 4701-4707, comparing the thermostability of phytases from basidiomycetes, in particular Peniophora lycii, Ceriporia sp., Trametes pubescens and Aspergillus niger; it appears that said enzymes have Tms (temperature at which the enzyme is 50% active) that are quite close, from 55° C. (Trametes pubescens) to 60° C. (Peniophora lycii) but have residual activities (percentage activity resulting from preincubation for 60 minutes at 80° C. in sodium acetate [0.1 M], pH 5.5) that vary widely, from 15% (Trametes pubescens) to 62% (Peniophora Lycii).
Many establishments have filed patent applications concerning said enzymes, in particular Danisco/Genencor (WO 2001/012792, Penicillium subtilis; WO 2003/038035, Trichoderma reesei; WO 2003/038111, Penicillium, Mumicola, Emericella, Fusarium), ABEnzymes/ROAL (EP 0 659 215, Aspergillus phytases produced by Trichoderma reesei), DSM/Roche (EP 0 684 313, Apergillus terreus, Aspergillus fumigatus, Aspergillus nidulans, Talaromaces thermophilus), BASF (WO 2003/102174, Aspergillus), Adisseo (WO 2003/054199, Penicillium), and Choongang Biotech Ltd. (WO 2005/056835, Penicillium oxalicum).
Several bacterial phytases have been described that originate from Bacillus subtilis (Paver and Jagannathan, 1982, Journal of Bacteriology 151:1102-1108), Pseudomonas (Cosgrove, 1970, Australian Journal of Biological Sciences 23:1207-1220), and Klebsiella. Several phytases originating from E. coli have been reported in the literature. Greiner et al, in Arch, Biochem. Biophys., 303, 107-113, 1993, purified and characterized a novel phytase of E. coli; others have been reported by Lim et al., 2000, Nat. Struct. Biol. 7: 108-113, Oshima et al., 1996, DNA Research, 3:137-155, Touati and Danchin, 1987, Biochimie, 69:215-221, Rodriguez et al., 2000, Arch. Biochem. Biophys., 382:105-112, Kretz, U.S. Pat. No. 5,876,997 from E. coli B, and appA by Dassa et al., 1990, J. Bacteriol. 172:5497-5500.
Mutants from E. coli phytase have been obtained by genetic engineering, resulting in enhanced thermostabilities and specific activities (Rodriguez et al, 2000, Arch. Biochem. Biophys., 382:105-112, Lanahan et al., 2003, US patent application 20030157646). However, none of those mutations could be used to produce sufficient of those enzymes of prokaryotic origin in eukaryotic production organisms.
The aim of the present invention is to provide a recombinant enzyme that is suitable for industrial processes to allow it to be used as a food additive, principally in animal feed.
In the application WO 2002/048332, using a BLAST analysis of bacterial genomes available at the date of the invention and using the appA gene from E. coli, Diversa identified a novel protein from Yersinia pestis having phytase activity. That protein had a remarkable feature, namely that it has a specific activity of 4400 U/mg. No other biochemical features of that protein were specified in that application. That application appears to demonstrate a high potential activity for phytase originating from bacteria from the Yersinia family.
On Oct. 20, 2005, the protein sequence with reference ZP—00832361 was added to the NCBI database. Said sequence is that of a hypothetical protein of Yersinia intermedia ATCC 29909 the corresponding nucleotide sequence for which is presented under reference numeral NZ_AALF01000052, region: 1889 . . . 3214. That protein sequence was obtained by translation of the corresponding nucleotide sequence originating from the complete sequence for the genome of the strain Yersinia intermedia ATCC 29909. Although the PRK10172 domain appears to predict a phytase activity, no experimental element accompanied that prediction.
This appears to have been confirmed by the isolation of a very similar phytase from a novel strain of Yersinia intermedia originating from a dirt sample from a glacier, denoted H-27 and presented in the application WO 2007/128160. The phytase derived from the H-27 strain is designated in the NCBI database with accession number AB195370.1 for the nucleotide sequence and DQ986462 for the protein sequence; it has 98% identity as regards the amino acids and 97% identity with the nucleotide sequence encoding the hypothetical phytase of Yersinia intermedia ATCC 29909.
The phytase of application WO 2007/128160 has a high specific activity of more than 3000 U/mg, of the same order as the specific activity recorded for Yersinia pestis in WO 2002/048332. In that application WO 2007/128160, the intrinsic biochemical characteristics of the protein are claimed, namely a molecular weight of 45.5 kDa, an optimum pH in the range 4.0 to 5.0, an optimum temperature in the range 50 to 60° C., a theoretical pI of 7.7, a specific activity of more than 3000 U/mg and a high resistance to pepsin and trypsin.